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In hypoxic and pseudohypoxic rodent models of pulmonary hypertension (PH), hypoxia-inducible factor (HIF) inhibition attenuates disease initiation. However, HIF activation alone, due to genetic alterations or use of inhibitors of prolyl hydroxylase domain (PHD) enzymes, has not been definitively shown to cause PH in humans, indicating the involvement of other mechanisms. Given the association between endothelial cell dysfunction and PH, the effects of pseudohypoxia and its underlying pathways were investigated in primary human lung endothelial cells. PHD2 silencing or inhibition, while activating HIF2α, induced apoptosis-resistance and IFN/STAT activation in endothelial cells, independent of HIF signaling. Mechanistically, PHD2 deficiency activated AKT and ERK, inhibited JNK, and reduced AIP1 (ASK1-interacting protein 1), all independent of HIF2α. Like PHD2, AIP1 silencing affected these same kinase pathways and produced a similar dysfunctional endothelial cell phenotype, which were partially reversed by AKT inhibition. Consistent with these in vitro findings, AIP1 protein levels in lung endothelial cells were decreased in Tie2-Cre/Phd2 knockout mice compared to wild-type controls. Lung vascular endothelial cells from patients with pulmonary arterial hypertension (PAH) showed IFN/STAT activation. Lung tissue from both SU5416/hypoxia PAH rats and PAH patients all showed AKT activation and dysregulated AIP1 expression. In conclusion, PHD2 deficiency in lung vascular endothelial cells drives an apoptosis-resistant and inflammatory phenotype, mediated by AKT activation and AIP1 loss independent of HIF signaling. Targeting these pathways, including PHD2, AKT, and AIP1, holds potential for developing new treatments for endothelial dysfunction in PH.
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We generated a replication-competent OC43 human seasonal coronavirus (CoV) expressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike in place of the native spike (rOC43-CoV2 S). This virus is highly attenuated relative to OC43 and SARS-CoV-2 in cultured cells and animals and is classified as a biosafety level 2 (BSL-2) agent by the NIH biosafety committee. Neutralization of rOC43-CoV2 S and SARS-CoV-2 by S-specific monoclonal antibodies and human sera is highly correlated, unlike recombinant vesicular stomatitis virus-CoV2 S. Single-dose immunization with rOC43-CoV2 S generates high levels of neutralizing antibodies against SARS-CoV-2 and fully protects human ACE2 transgenic mice from SARS-CoV-2 lethal challenge, despite nondetectable replication in respiratory and nonrespiratory organs. rOC43-CoV2 S induces S-specific serum and airway mucosal immunoglobulin A and IgG responses in rhesus macaques. rOC43-CoV2 S has enormous value as a BSL-2 agent to measure S-specific antibodies in the context of a bona fide CoV and is a candidate live attenuated SARS-CoV-2 mucosal vaccine that preferentially replicates in the upper airway.
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Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , Testes de Neutralização , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Humanos , Anticorpos Neutralizantes/imunologia , Camundongos , COVID-19/imunologia , COVID-19/virologia , COVID-19/prevenção & controle , Anticorpos Antivirais/imunologia , Testes de Neutralização/métodos , Camundongos Transgênicos , Coronavirus Humano OC43/imunologia , Coronavirus Humano OC43/genética , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/imunologia , Chlorocebus aethiops , Células Vero , Macaca mulattaRESUMO
Cellular therapies with cardiomyocytes produced from induced pluripotent stem cells (iPSC-CMs) offer a potential route to cardiac regeneration as a treatment for chronic ischemic heart disease. Here, we report successful long-term engraftment and in vivo maturation of autologous iPSC-CMs in two rhesus macaques with small, subclinical chronic myocardial infarctions, all without immunosuppression. Longitudinal positron emission tomography imaging using the sodium/iodide symporter (NIS) reporter gene revealed stable grafts for over 6 and 12 months, with no teratoma formation. Histological analyses suggested capability of the transplanted iPSC-CMs to mature and integrate with endogenous myocardium, with no sign of immune cell infiltration or rejection. By contrast, allogeneic iPSC-CMs were rejected within 8 weeks of transplantation. This study provides the longest-term safety and maturation data to date in any large animal model, addresses concerns regarding neoantigen immunoreactivity of autologous iPSC therapies, and suggests that autologous iPSC-CMs would similarly engraft and mature in human hearts.
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Células-Tronco Pluripotentes Induzidas , Macaca mulatta , Miócitos Cardíacos , Animais , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Diferenciação Celular , Humanos , Transplante Autólogo , Tomografia por Emissão de Pósitrons , Fatores de Tempo , Infarto do Miocárdio/terapia , Infarto do Miocárdio/patologiaRESUMO
Neonates and young infants are known to have limited responses to pediatric vaccines due to reduced germinal center formation. Extended vaccine antigen dosing was previously shown to expand germinal center formation and improve humoral responses in adult mice. We report that sustained antigen delivery through sequential dosing overcomes neonatal limitations to form germinal center reactions and improves humoral immunity. Thus, vaccine strategies that extend the release of vaccine antigens may reduce the number of doses, and time needed, to achieve protective immunity in neonates and young infants.
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Hepatitis E virus (HEV) causes self-limited acute hepatitis in immunocompetent individuals and can establish chronic infection in solid organ transplant recipients taking immunosuppressive drugs. A well characterized small animal model is needed to understand HEV pathogenesis. In this study, we established a robust model to study acute and persistent HEV infection using Mongolian gerbils (Meriones unguiculatus) with or without immunosuppression. Gerbils were implanted subcutaneously with continuous release tacrolimus pellet to induce immunosuppression. Gerbils with or without tacrolimus treatment were inoculated with HEV intraperitoneally. Viremia, fecal virus shedding, serum antibody and ALT levels, liver histopathological lesions, hepatocyte apoptosis, and liver macrophage distribution were assessed. Mild to moderate self-limited hepatitis and IgM and IgG antibody responses against HEV ORF2 were observed in immunocompetent gerbils. Levels of HEV-specific IgM responses were higher and lasted longer in immunocompetent gerbils with higher peak viremia. Persistent viremia and fecal virus shedding with either weak, or absent HEV antibody levels were seen in immunosuppressed gerbils. Following HEV infection, serum ALT levels were increased, with lower and delayed peaks observed in immunosuppressed compared to immunocompetent gerbils. In immunocompetent gerbils, foci of apoptotic hepatocytes were detected that were distributed with inflammatory infiltrates containing CD68+ macrophages. However, these foci were absent in immunosuppressed gerbils. The immunosuppressed gerbils showed no inflammation with no increase in CD68+ macrophages despite high virus replication in liver. Our findings suggest adaptive immune responses are necessary for inducing hepatocyte apoptosis, CD68+ macrophage recruitment, and inflammatory cell infiltration in response to HEV infection. Our studies show that Mongolian gerbils provide a promising model to study pathogenesis during acute and persistent HEV infection.
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Vírus da Hepatite E , Hepatite E , Animais , Humanos , Vírus da Hepatite E/genética , Gerbillinae , Tacrolimo , Viremia , GenótipoRESUMO
Because of rapid emergence and circulation of the SARS-CoV-2 variants, especially Omicron which shows increased transmissibility and resistant to antibodies, there is an urgent need to develop novel therapeutic drugs to treat COVID-19. In this study we developed an in vitro cellular model to explore the regulation of ACE2 expression and its correlation with ACE2-mediated viral entry. We examined ACE2 expression in a variety of human cell lines, some of which are commonly used to study SARS-CoV-2. Using the developed model, we identified a number of inhibitors which reduced ACE2 protein expression. The greatest reduction of ACE2 expression was observed when CK869, an inhibitor of the actin-related protein 2/3 (ARP2/3) complex, was combined with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), an inhibitor of sodium-hydrogen exchangers (NHEs), after treatment for 24 h. Using pseudotyped lentivirus expressing the SARS-CoV-2 full-length spike protein, we found that ACE2-dependent viral entry was inhibited in CK869 + EIPA-treated Calu-3 and MDA-MB-468 cells. This study provides an in vitro model that can be used for the screening of novel therapeutic candidates that may be warranted for further pre-clinical and clinical studies on COVID-19 countermeasures.
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3D bioprinting technology has gained increased attention in the regenerative medicine and tissue engineering communities over the past decade with their attempts to create functional living tissues and organsde novo. While tissues such as skin, bone, and cartilage have been successfully fabricated using 3D bioprinting, there are still many technical and process driven challenges that must be overcome before a complete tissue engineered solution is realized. Although there may never be a single adopted bioprinting process in the scientific community, adherence to optimized bioprinting protocols could reduce variability and improve precision with the goal of ensuring high quality printed constructs. Here, we report on the bioprinting of a gelatin-alginate-collagen bioink containing human mesenchymal stromal cells (hMSCs) which has been optimized to ensure printing consistency and reliability. The study consists of three phases: a pre-printing phase which focuses on bioink characterization; a printing phase which focuses on bioink extrudability/printability, construct stability, and printing accuracy; and a post-processing phase which focuses on the homogeneity and bioactivity of the encapsulated hMSC printed constructs. The results showed that eight identical constructs containing hMSCs could be reliably and accurately printed into stable cross-hatched structures with a single material preparation, and that batch-to-batch consistency was accurately maintained across all preparations. Analysis of the proliferation, morphology, and differentiation of encapsulated hMSCs within the printed constructs showed that cells were able to form large,interconnected colonies and were capable of robust adipogenic differentiation within 14 d of culturing.
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Gelatina , Células-Tronco Mesenquimais , Humanos , Alginatos , Reprodutibilidade dos Testes , ColágenoRESUMO
Leishmaniasis is a vector-borne parasitic disease transmitted through the bite of a sand fly with no available vaccine for humans. Recently, we have developed a live attenuated Leishmania major centrin gene-deleted parasite strain (LmCen-/- ) that induced protection against homologous and heterologous challenges. We demonstrated that the protection is mediated by IFN (Interferon) γ-secreting CD4+ T-effector cells and multifunctional T cells, which is analogous to leishmanization. In addition, in a leishmanization model, skin tissue-resident memory T (TRM) cells were also shown to be crucial for host protection. In this study, we evaluated the generation and function of skin TRM cells following immunization with LmCen-/- parasites and compared those with leishmanization. We show that immunization with LmCen-/- generated skin CD4+ TRM cells and is supported by the induction of cytokines and chemokines essential for their production and survival similar to leishmanization. Following challenge with wild-type L. major, TRM cells specific to L. major were rapidly recruited and proliferated at the site of infection in the immunized mice. Furthermore, upon challenge, CD4+ TRM cells induce higher levels of IFNγ and Granzyme B in the immunized and leishmanized mice than in non-immunized mice. Taken together, our studies demonstrate that the genetically modified live attenuated LmCen-/- vaccine generates functional CD4+ skin TRM cells, similar to leishmanization, that may play a crucial role in host protection along with effector T cells as shown in our previous study.
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Leishmania major , Vacinas contra Leishmaniose , Parasitos , Animais , Imunidade , Interferon gama , Vacinas contra Leishmaniose/genética , Células T de Memória , Camundongos , Pele , Combinação Trimetoprima e SulfametoxazolRESUMO
BACKGROUND: Neutrophils are involved in the initial host responses to pathogens. Neutrophils can activate T cell responses either independently or through indirect involvement of Dendritic cells (DCs). Recently we have demonstrated direct neutrophil-T cell interactions that initiate adaptive immune responses following immunization with live attenuated Leishmania donovani centrin deleted parasite vaccine (LdCen-/-). However, neutrophil-DC interactions in T cell priming in vaccine immunity in general are not known. In this study we evaluated the interaction between neutrophils and DCs during LdCen-/- infection and compared with wild type parasite (LdWT) both in vitro and in vivo. METHODOLOGY/FINDINGS: LdCen-/- parasite induced increased expression of CCL3 in neutrophils caused higher recruitment of DCs capable of inducing a strong proinflammatory response and elevated co-stimulatory molecule expression compared to LdWT infection. To further illustrate neutrophil-DCs interactions in vivo, we infected LYS-eGFP mice with red fluorescent LdWT/LdCen-/- parasites and sort selected DCs that engulfed the neutrophil containing parasites or DCs that acquired the parasites directly in the ear draining lymph nodes (dLN) 5d post infection. The DCs predominantly acquired the parasites by phagocytosing infected neutrophils. Specifically, DCs containing LdCen-/- parasitized neutrophils exhibited a proinflammatory phenotype, increased expression of costimulatory molecules and initiated higher CD4+T cell priming ex-vivo. Notably, potent DC activation occurred when LdCen-/- parasites were acquired indirectly via engulfment of parasitized neutrophils compared to direct engulfment of LdCen-/- parasites by DCs. Neutrophil depletion in LdCen-/- infected mice significantly abrogated expression of CCL3 resulting in decreased DC recruitment in ear dLN. This event led to poor CD4+Th1 cell priming ex vivo that correlated with attenuated Tbet expression in ear dLN derived CD4+ T cells in vivo. CONCLUSIONS: Collectively, LdCen-/- containing neutrophils phagocytized by DC markedly influence the phenotype and antigen presenting capacity of DCs early on and thus play an immune-regulatory role in shaping vaccine induced host protective response.
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Leishmania donovani , Vacinas contra Leishmaniose , Leishmaniose Visceral , Animais , Comunicação Celular , Células Dendríticas , Leishmania donovani/fisiologia , Leishmaniose Visceral/parasitologia , Camundongos , Neutrófilos , Vacinas AtenuadasRESUMO
Purpose: Whole slide imaging (WSI) scanners produce tissue slide images with a large field of view and a high resolution for pathologists to use in diagnoses. Color performance tests of these color imaging devices are necessary and can use stained tissue slides if the color truth is established using a hyperspectral imaging microscopy system (HIMS). The purpose of this study was to estimate the reproducibility uncertainty of CIELAB coordinates for a reference tissue slide measured by both the HIMS and a WSI scanner. Approach: We compared the color performances of the WSI scanner to those of the reference established by the HIMS using the International Commission on Illumination (Commission Internationale de l'Éclairage, or CIE) 1976 Δ E a b * color difference with the just noticeable color difference (JNCD, Δ E a b * ≤ 2 ), and the results from the overlap of the CIELAB coordinates' uncertainty within the error bar, with a coverage factor k = 2 . The reported uncertainty results from measurements and image registration uncertainties. Results: For the blank area common to the HIMS and the WSI average images, the color agreement was higher using the JNCD condition versus the CIELAB uncertainty overlap criterion (82% and 20% of the pixels in the images, respectively). This difference is explained by the fact that numerous pixels have CIELAB coordinates near one another but corresponding to CIELAB uncertainty values small enough not to overlap. In the colored area of the images, the JNCD condition was met for 0.19% of the pixels in the images, compared with 4.3% for the CIELAB uncertainty overlap criterion. Conclusions: The distribution of uncertainties on the CIELAB coordinates was broader for the HIMS compared with the WSI scanner. The WSI scanner had a systemic error in the color reproduction, which pointed to a potential inadequate color calibration of this device.
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Despite their exceptional capacity for transgene delivery ex vivo, lentiviral (LV) vectors have been slow to demonstrate clinical utility in the context of in vivo applications. Unresolved safety concerns related to broad LV vector tropism have limited LV vectors to ex vivo applications. Here, we report on a novel LV vector-pseudotyping strategy involving envelope glycoproteins of Tupaia paramyxovirus (TPMV) engineered to specifically target human cell-surface receptors. LV vectors pseudotyped with the TPMV hemagglutinin (H) protein bearing the interleukin (IL)-13 ligand in concert with the TPMV fusion (F) protein allowed efficient transduction of cells expressing the human IL-13 receptor alpha 2 (IL-13Rα2). Immunodeficient mice bearing orthotopically implanted human IL-13Rα2 expressing NCI-H1299 non-small cell lung cancer cells were injected intravenously with a single dose of LV vector pseudotyped with the TPMV H-IL-13 glycoprotein. Vector biodistribution was monitored using bioluminescence imaging of firefly luciferase transgene expression, revealing specific transduction of tumor tissue. A quantitative droplet digital PCR (ddPCR) analysis of lung tissue samples revealed a >15-fold increase in the tumor transduction in mice treated with LV vectors displaying IL-13 relative to those without IL-13. Our results show that TPMV envelope glycoproteins can be equipped with ligands to develop targeted LV vectors for in vivo applications.
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Malaria vaccines that disrupt the Plasmodium life cycle in mosquitoes and reduce parasite transmission in endemic areas are termed transmission-blocking vaccines (TBVs). Despite decades of research, there are only a few Plasmodium falciparum antigens that indisputably and reproducibly demonstrate transmission-blocking immunity. So far, only two TBV candidates have advanced to phase 1/2 clinical testing with limited success. By applying an unbiased transcriptomics-based approach, we have identified Pf77 and male development gene 1 (PfMDV-1) as two P. falciparum TBV antigens that, upon immunization, induced antibodies that caused reductions in oocyst counts in Anopheles mosquito midguts in a standard membrane feeding assay. In-depth studies were performed to characterize the genetic diversity of, stage-specific expression by, and natural immunity to these two molecules to evaluate their suitability as TBV candidates. Pf77 and PfMDV-1 display limited antigenic polymorphism, are pan-developmentally expressed within the parasite, and induce naturally occurring antibodies in Ghanaian adults, which raises the prospect of natural boosting of vaccine-induced immune response in endemic regions. Together, these biological properties suggest that Pf77 and PfMDV-1 may warrant further investigation as TBV candidates.
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Vacinas Antimaláricas , Malária Falciparum , Animais , Anticorpos Antiprotozoários , Antígenos de Protozoários/genética , Gana , Malária Falciparum/prevenção & controle , Masculino , Plasmodium falciparumRESUMO
Interferonopathies, interferon (IFN)-α/ß therapy, and caveolin-1 (CAV1) loss-of-function have all been associated with pulmonary arterial hypertension (PAH). Here, CAV1-silenced primary human pulmonary artery endothelial cells (PAECs) were proliferative and hypermigratory, with reduced cytoskeletal stress fibers. Signal transducers and activators of transcription (STAT) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) were both constitutively activated in these cells, resulting in a type I IFN-biased inflammatory signature. Cav1-/- mice that spontaneously develop pulmonary hypertension were found to have STAT1 and AKT activation in lung homogenates and increased circulating levels of CXCL10, a hallmark of IFN-mediated inflammation. PAH patients with CAV1 mutations also had elevated serum CXCL10 levels and their fibroblasts mirrored phenotypic and molecular features of CAV1-deficient PAECs. Moreover, immunofluorescence staining revealed endothelial CAV1 loss and STAT1 activation in the pulmonary arterioles of patients with idiopathic PAH, suggesting that this paradigm might not be limited to rare CAV1 frameshift mutations. While blocking JAK/STAT or AKT rescued aspects of CAV1 loss, only AKT inhibitors suppressed activation of both signaling pathways simultaneously. Silencing endothelial nitric oxide synthase (NOS3) prevented STAT1 and AKT activation induced by CAV1 loss, implicating CAV1/NOS3 uncoupling and NOS3 dysregulation in the inflammatory phenotype. Exogenous IFN reduced CAV1 expression, activated STAT1 and AKT, and altered the cytoskeleton of PAECs, implicating these mechanisms in PAH associated with autoimmune and autoinflammatory diseases, as well as IFN therapy. CAV1 insufficiency elicits an IFN inflammatory response that results in a dysfunctional endothelial cell phenotype and targeting this pathway may reduce pathologic vascular remodeling in PAH.
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Caveolina 1/genética , Endotélio Vascular/metabolismo , Hipertensão Pulmonar/metabolismo , Interferon Tipo I/metabolismo , Animais , Células Cultivadas , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Inativação Gênica , Humanos , Hipertensão Pulmonar/fisiopatologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição STAT1/metabolismo , Transdução de SinaisRESUMO
Pegylated interferon-α (PEG-IFN-α), where IFN-α is attached to polyethylene glycol (PEG), is an approved treatment for chronic hepatitis B virus (HBV) infection, a disease that causes liver-related morbidity and mortality in 257 million people worldwide. It is unknown why only a minority of patients respond to PEG-IFN-α. Using sequential blood samples and liver biopsies of patients with chronic HBV infection before, during, and after PEG-IFN-α treatment, we find that patients with early natural killer (NK) cell activation after PEG-IFN-α injection experienced greater liver inflammation, lysis of HBV-infected hepatocytes, and hepatitis B surface antigen (HBsAg) decline than those without. NK cell activation was associated with induction of interferon-stimulated genes and determined by PEG-IFN-α pharmacokinetics. Patients with delayed increases in PEG-IFN-α concentrations had greater amounts of PEG-specific immunoglobulin M (IgM) immune complexes in the blood and more PEG and IgM detected in the liver than patients with rapid increase in PEG-IFN-α concentration. This was associated with reduced NK cell activation. These results indicate that the immunomodulatory functions of PEG-IFN-α, particularly activation of NK cells, play a pivotal role in the response to treatment and further demonstrate that these functions are affected by PEG-IFN-α pharmacokinetics. Accelerated clearance of antibody-complexed pegylated drugs by Kupffer cells may be important beyond the field of HBV therapeutics. Thus, these findings may contribute to improving the efficacy of pegylated drugs that are now being developed for other chronic diseases and cancer.
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Vírus da Hepatite B , Hepatite B Crônica , Antivirais/uso terapêutico , Hepatite B Crônica/tratamento farmacológico , Humanos , Células Matadoras Naturais , Células de Kupffer , Polietilenoglicóis , Proteínas Recombinantes/uso terapêutico , Resultado do TratamentoRESUMO
Anthracyclines are a class of chemotherapy drugs that are highly effective for the treatment of human cancers, but their clinical use is limited by associated dose-dependent cardiotoxicity. The precise mechanisms by which individual anthracycline induces cardiotoxicity are not fully understood. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are emerging as a physiologically relevant model to assess drugs cardiotoxicity. Here, we describe an assay platform by coupling hiPSC-CMs and impedance measurement, which allows real-time monitoring of cardiomyocyte cellular index, beating amplitude, and beating rate. Using this approach, we have performed comparative studies on a panel of four anthracycline drugs (doxorubicin, epirubicin, idarubicin, and daunorubicin) which share a high degree of structural similarity but are associated with distinct cardiotoxicity profiles and maximum cumulative dose limits. Notably, results from our hiPSC-CMs impedance model (dose-dependent responses and EC50 values) agree well with the recommended clinical dose limits for these drugs. Using time-lapse imaging and RNAseq, we found that the differences in anthracycline cardiotoxicity are closely linked to extent of cardiomyocyte uptake and magnitude of activation/inhibition of several cellular pathways such as death receptor signaling, ROS production, and dysregulation of calcium signaling. The results provide molecular insights into anthracycline cardiac interactions and offer a novel assay system to more robustly assess potential cardiotoxicity during drug development.
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Antraciclinas/efeitos adversos , Antibióticos Antineoplásicos/efeitos adversos , Cardiotoxicidade/etiologia , Miócitos Cardíacos/efeitos dos fármacos , Bioensaio/métodos , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Impedância Elétrica , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Microscopia Intravital/métodos , Miócitos Cardíacos/fisiologia , Estresse Oxidativo/efeitos dos fármacos , RNA-Seq , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Imagem com Lapso de TempoRESUMO
Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.
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COVID-19/metabolismo , Lentivirus/metabolismo , Testes de Neutralização/métodos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
No licensed vaccine exists against visceral leishmaniasis (VL), a disease caused by the Leishmania donovani parasite. We have previously reported both macrophages and dendritic cells play important role in the protection induced by a live attenuated centrin gene-deleted L. donovani (LdCen-/- ) parasite vaccine. The role of neutrophils in orchestrating the initial innate response to pathogens is widely recognized. To investigate the early interaction of LdCen-/- with neutrophils, we immunized mice intradermally in the ear pinna with LdCen-/- Compared with LdWT infection, LdCen-/- parasites induced higher recruitment of neutrophils to the ear dermis and ear draining lymph nodes (dLN) as early as 6-18 h after immunization, which were predominantly proinflammatory in nature. Neutrophils from ear dLN of LdCen-/- -immunized mice exhibited heightened expression of costimulatory molecules and attenuated expression of coinhibitory molecules necessary for higher T cell activation. Further phenotypic characterization revealed heterogeneous neutrophil populations containing Nα and Nß subtypes in the ear dLN. Of the two, the parasitized Nα subset from LdCen-/- -immunized mice exhibited much stronger Ag-specific CD4+ T cell proliferation ex vivo. Adoptive transfer of neutrophils bearing LdCen-/- parasites induced an increased Th1 response in naive mice. Importantly, neutrophil depletion significantly abrogated Ag-specific CD4+ T cell proliferation in LdCen-/- -immunized mice and impaired protection against virulent challenge. Conversely, replenishing of neutrophils significantly restored the LdCen-/- -induced host-protective response. These results suggest that neutrophils are indispensable for protective immunity induced by LdCen-/- parasite vaccine.
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Leishmania donovani/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/prevenção & controle , Ativação Linfocitária , Infiltração de Neutrófilos , Neutrófilos/imunologia , Células Th1/imunologia , Animais , Feminino , Leishmania donovani/genética , Vacinas contra Leishmaniose/genética , Leishmaniose Visceral/genética , Leishmaniose Visceral/imunologia , Camundongos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologiaRESUMO
CD138 (syndecan 1), a member of the heparan-sulfate proteoglycan family, regulates diverse biological responses by interacting with chemokines, cytokines, growth factors, and adhesion molecules. Expression of CD138 has been detected on T cells from both healthy and sick mice mimicking systemic lupus erythematosus (SLE) disease. However, the characteristics and the role of CD138+ T cells in SLE pathogenesis remain largely unknown. We analyzed the lupus-prone MRL/Lpr mice and the control MRL/MpJ strain as well as the common laboratory strains Balb/c, and C57BL/6 for CD138-expression and found that only the MRL/Lpr strain harbored TCRß+CD138+ cells in various organs. The frequency of TCRß+CD138+ cells progressively expanded in MRL/Lpr mice with age and correlated with disease severity. Majority of the TCRß+CD138+ cells were CD4 and CD8 double-negative and 20% were CD4. At least a portion of TCRß+CD138+ cells originated from CD4+ cells because substantial number of CD4+TCRß+CD138- cells expressed CD138 after in vitro cultivation. Compared to TCRß+CD138- cells, TCRß+CD138+ cells exhibited central memory (Tcm) phenotype with reduced ability to proliferate and produce the cytokines IFNγ and IL-17. When co-cultured with B cells, the ability of TCRß+CD138+ cells to promote plasma cell formation and autoreactive antibody production was dependent on the presence of autoantigen, CD4 co-receptor expression and cell-to-cell contact. Surprisingly, adoptively transferred TCRß+CD138+ T cells slowed down disease progression in young recipient MRL/Lpr mice but had the opposite effect when DNA was co-administered with TCRß+CD138+ T cells or when TCRß+CD138+ cells were transferred to older MRL/Lpr mice with established disease. Thus, CD138-expressing T cells with Tcm phenotype enhance disease progression in SLE by rapidly activating autoreactive B cells when self-antigens are exposed to the immune system.
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Suscetibilidade a Doenças , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/metabolismo , Sindecana-1/genética , Sindecana-1/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transferência Adotiva , Animais , Anticorpos Antinucleares/imunologia , Autoimunidade , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , Modelos Animais de Doenças , Citometria de Fluxo , Expressão Gênica , Lúpus Eritematoso Sistêmico/patologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos MRL lpr , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Production of IFN-γ is a key innate immune mechanism that limits replication of intracellular bacteria such as Francisella tularensis (Ft) until adaptive immune responses develop. Previously, we demonstrated that the host cell types responsible for IFN-γ production in response to murine Francisella infection include not only natural killer (NK) and T cells, but also a variety of myeloid cells. However, production of IFN-γ by mouse dendritic cells (DC) is controversial. Here, we directly demonstrated substantial production of IFN-γ by DC, as well as hybrid NK-DC, from LVS-infected wild type C57BL/6 or Rag1 knockout mice. We demonstrated that the numbers of conventional DC producing IFN-γ increased progressively over the course of 8 days of LVS infection. In contrast, the numbers of conventional NK cells producing IFN-γ, which represented about 40% of non-B/T IFN-γ-producing cells, peaked at day 4 after LVS infection and declined thereafter. This pattern was similar to that of hybrid NK-DC. To further confirm IFN-γ production by infected cells, DC and neutrophils were sorted from naïve and LVS-infected mice and analyzed for gene expression. Quantification of LVS by PCR revealed the presence of Ft DNA not only in macrophages, but also in highly purified, IFN-γ producing DC and neutrophils. Finally, production of IFN-γ by infected DC was confirmed by immunohistochemistry and confocal microscopy. Notably, IFN-γ production patterns similar to those in wild type mice were observed in cells derived from LVS-infected TLR2, TLR4, and TLR2xTLR9 knockout (KO) mice, but not from MyD88 KO mice. Taken together, these studies demonstrate the pivotal roles of DC and MyD88 in IFN-γ production and in initiating innate immune responses to this intracellular bacterium.
Assuntos
Interferon gama/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Receptores Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Francisella tularensis/imunologia , Imunidade Inata/imunologia , Células Matadoras Naturais/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Neutrófilos/metabolismo , Baço/metabolismo , Linfócitos T/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/imunologia , Tularemia/microbiologiaRESUMO
Babesia microti is an intraerythrocytic parasite and the primary causative agent of human babesiosis. It is transmitted by Ixodes ticks, transfusion of blood and blood products, organ donation, and perinatally. Despite its global public health impact, limited progress has been made to identify and characterize immunodominant B. microti antigens for diagnostic and vaccine use. Using genome-wide immunoscreening, we identified 56 B. microti antigens, including some previously uncharacterized antigens. Thirty of the most immunodominant B. microti antigens were expressed as recombinant proteins in E. coli. Among these, the combined use of two novel antigens and one previously described antigen provided 96% sensitivity and 100% specificity in identifying B. microti antibody containing sera in an ELISA. Using extensive computational sequence and bioinformatics analyses and cellular localization studies, we have clarified the domain architectures, potential biological functions, and evolutionary relationships of the most immunodominant B. microti antigens. Notably, we found that the BMN-family antigens are not monophyletic as currently annotated, but rather can be categorized into two evolutionary unrelated groups of BMN proteins respectively defined by two structurally distinct classes of extracellular domains. Our studies have enhanced the repertoire of immunodominant B. microti antigens, and assigned potential biological function to these antigens, which can be evaluated to develop novel assays and candidate vaccines.